Abstract
Corolla color in Gentiana lutea L. exhibits a yellow/orange variation. We previously demonstrated that the orange petal color of G. lutea L. var. aurantiaca is predominantly caused by newly synthesized pelargonidin glycosides that confer a reddish hue to the yellow background color, derived from the carotenoids. However, the anthocyanin molecules of these pelargonidin glycosides are not yet fully identified and characterized. Here, we investigated the regulation, content and type of anthocyanins determining the petal coloration of the orange-flowered G. lutea L. var. aurantiaca. Anthocyanins from the petals of G. lutea L. var. aurantiaca were characterized and quantified by HPLC-ESI-MS/MS (High-performance liquid chromatography-electrospray ionization-tandem mass spectrometry) coupled with a diode array detector in flowers at three different stages of development (S1, S3 and S5). Eleven pelargonidin derivatives were identified in the petals of G. lutea L. var. aurantiaca for the first time, but quantitative and qualitative differences were observed at each developmental stage. The highest levels of these pelargonidin derivatives were reached at the fully open flower stage (S5) where all anthocyanins were detected. In contrast, not all the anthocyanins were detected at the budlet stage (S1) and mature bud stage (S3) and those corresponded to more complex pelargonidin derivatives. The major pelargonidin derivatives found at all the stages were pelargonidin 3-O-glucoside, pelargonidin 3,5-O-diglucoside and pelargonidin 3-O-rutinoside. Furthermore, the expression of DFR (dihydroflavonol 4-reductase), ANS (anthocyanidin synthase), 3GT (UDP-glucose:flavonoid 3-O-glucosyltransferase), 5GT (UDP-glucose:flavonoid 5-O-glucosyltransferase) and 5AT (anthocyanin 5-aromatic acyltransferase) genes was analyzed in the petals of three developmental stages, showing that the expression level of DFR, ANS and 3GT parallels the accumulation of the pelargonidin glucosides. Overall, this study enhances the knowledge of the biochemical basis of flower coloration in Gentiana species, and lays a foundation for breeding of flower color and genetic variation studies on Gentiana varieties.
Highlights
Polymorphism in flower color has been associated with the preferences of pollinators for certain colorations, primarily due to the ability of pollinators to perceive and distinguish among different hues [1,2,3]
The genes encoding for enzymes of the anthocyanin pathway are grouped in two classes (Fig 1): early biosynthetic genes, which are common to other flavonoids, encoding enzymes including chalcone synthase (CHS) which is responsible for the formation of naringenin chalcone from 4-coumaroyl CoA and malonyl CoA substrates, chalcone isomerase (CHI) which generates flavones, flavanone 3-hydroxylase (F3H) which produces dihydrokaempferol, flavonoid 30-hydroxylase (F30H) and flavonoid 30,50-hydroxylase (F3050H) which generate dihydroquercetin and dihydromyricetin respectively; and late biosynthetic genes, specific to the anthocyanin pathway, encoding enzymes including dihydroflavonol 4-reductase (DFR) and anthocyanidin synthase (ANS) [5,6,7]
Peak 6 had a molecular ion m/z 1079.30284, and four fragment ions: m/z 932.23871 and m/z 771.21368 indicated the loss of coumaroylglucoside; m/z 771.21368 and m/z 433.11273 indicated the loss of feruloylglucoside, which resulted in pelargonidin glucoside; and m/z 271.05972, corresponding to pelargonidin (Fig 2J)
Summary
Polymorphism in flower color has been associated with the preferences of pollinators for certain colorations, primarily due to the ability of pollinators to perceive and distinguish among different hues [1,2,3]. The glycosylation reactions provide sugar residues for decoration with acyl groups by the activity of anthocyanin acyltransferase, which can incorporate aromatic acids such as p-coumaric, caffeic, ferulic, sinapic, gallic or p-hydroxybenzoic acids, and/or aliphatic acids including malonic, malic, acetic, succinic or oxalic acids. Other modification occurs, such as acylation of the glucose moiety at the 5-position of anthocyanin catalyzed by anthocyanin 5-aromatic acyltransferase (5AT) [12] Both glycosyltransferases and acyltransferases contribute to the extensive range of natural anthocyanins identified to date, which differ in their side chain decorations, affecting color and increasing pigment stability [6]
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